• 약학회지
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• 제34권1호
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• pp.11-14
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• 1990

$NH_2-terminal$ amino acid sequence analysis of monoclonal antibody is very important to identify gene family and diversities of antigen-antibody recognition. When we used the PVDF (Polyvinylidene difluoride) membrane blotting method, we could easily analyze $NH_2-terminal$ sequence of monoclonal-antibody which specifically binds to phosphatidylinositol 4,5-biphosphate. PVDF membrane is an ideal solid-phase support for sequence analysis, especially when used with electroblotting method. This method is superior to continual method and will be applied to the sequence analysis of picomole quantities of proteins by gel electrophoresis.

• Applied Biological Chemistry
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• 제16권3호
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• pp.112-117
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• 1973

The very low density apolipoproteins were separated by a newly developed method of isoelectric focusing in a narrow pH gradient. Four polypeptides were isolated that differed from the major proteins of the high density or low density lipoproteins. Three of these proteins had indistinguishable amino acid compositions, but different isoelectric points, COOH-terminal alanine, no isoleucine, cysteine or cystine. Two of these polypeptides had $NH_2-terminal$ serine. The polymorphism of apolipoprotein-Ala, so designated from the COOH-terminal residue, was related to sialic acid content; one form contained 2 moles of sialic acid per mole of protein, the second, 1 mole of protein, and the third, no sialic acid. The fourth polypeptide had an amino acid composition different from the first three polypeptides and from other polypetides obtained from very low density lipoprotein. This polypeptide had $NH_2-terminal$ threonine, COOH-terminal resistant to carboxypeptidase A, no histidine, cysteine, cystine or sialic acid. These four polypeptides constituted approx. 40% of the total protein in very low density lipoprotein.

• 공업화학
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• 제31권5호
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• pp.560-567
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• 2020

본 연구는 다양한 산업공정의 배가스 중 대표적인 활성저하 물질로 알려진 알칼리 금속[Na(Sodium)과 K(Potassium)]이 V/W/TiO₂ 촉매의 NH₃-SCR 반응에 미치는 영향을 확인 하였다. 이에 따른 활성 저하 원인을 규명하고자 NO, NH₃-TPD, DRIFT, H₂-TPR 분석을 수행 하였다. 그 결과, 각 알칼리 금속은 촉매 피독으로 작용하여 NH₃ 흡착양이 저하되고, Na과 K은 촉매의 반응 활성에 기여하는 L산점과 B산점을 감소시켜 SCR 반응을 저하시킨다. H₂-TPR 분석을 통하여 알칼리 금속은 V-O-V (bridge oxygen bond)와 V=O (terminal bond)의 환원 온도에 영향을 끼쳐, 환원 온도가 고온으로 올라가기 때문에 활성 저하 원인으로 판단된다.

• Animal cells and systems
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• 제1권2호
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• pp.323-328
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• 1997

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-9, was purified by conventional chromatographic procedures using $^{125}l$-labeled ubiquitin-${\alpha}$NH-MHISPPEPESEEEEE HYC (Ub-PESTc) as a substrate. The purified enzyme behaved as a 27-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Lice other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub-aldehyde. In addition to Ub-PESTc, UCH-9 hydrolyzed Ub-aNH-protein extensions, including Ub-${\alpha}NH$-carboxyl extension protein of 80 amino acids and Ubo-${\alpha}NH$-dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono-Ub-${\varepsilon}NH$-protein conjugates and from branched poly-Ub chains that are ligated to proteins through ${\varepsilon}NH$-isopeptide bonds. This enzyme neither could hydrolyze poly-His-tagged di-Ub. These results suggest that UCH-9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.

• Archives of Pharmacal Research
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• 제20권6호
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• pp.590-596
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• 1997

The NADH reductase component of the steroid 9.alpha.-hydroxylase from Mycobacterium fortuitum was purified to homogeneity. Recovery of the enzyme from the 50-60% ammonium sulfate saturated fraction was 49%, with a purification factor of 100-fold. The NADH reductase has a relative molecular of 60 KDa as determined by SDS-PAGE. The absorption maxima at 410 and 450 nm indicate the presence of iron-sulfur group and flavin. These prosthetic groups seemed to function as redox groups that transfer electrons from NADH to the following protein. The $K_M$ value for NADH as substrate was $68{\mu}M$. The $NH_2$-terminal amino acid sequence of the reductase was determined as Met-Asp-Ala-Ile-Thr-Asn-Val-Pro-Leu-Pro-Ala-Asn-Glu-Pro-Val-His-Asp-Tyr-Ala-Thr. This sequence does not show a homology with the $NH_2$ -terminal sequences reported for the reductase component of other monooxygenases, suggesting that the NADH reductase component of the steroid 9.alpha.-hydroxylase system is novel.

• 한국식품과학회지
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• 제31권2호
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• pp.482-487
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• 1999

과요오드산으로 산화시킨 당을 유리아미노산과 반응시켜서 ${\alpha}-NH_2$기와 펩티딜리신의 ${\varepsilon}-NH_2$기와 반응하여 결합한 것을 확인하였다. 그래서, 제조한 과요드산-산화당은 단백질 분자 표면의 리신의 ${\varepsilon}-NH_2$기와 Schiff 염기 반응으로 공유결합하여 당단백질을 만드는 것으로 볼 수 있다. 과요드산 산화당으로 변형한 고구마 ${\beta}$-아밀라아제는 C, H는 증가, N은 감소하였다. N말단의 ${\alpha}-NH_2$기는 70% (pentamer), 73% (monomer), ${\varepsilon}-NH_2$기는 33% (pentamer), 26% (monomer)가 산화당과 반응하였다. 페놀-황산법에 의한 총당 정량과 DNP 법으로 분석한 결과, $IO_4$-산화말토헥사오스는 고구마 ${\beta}$-아밀라아제 1몰당 6몰이 결합된 것으로 나타났고, 고구마 ${\beta}$-아밀라아제에 결합된 산화가용성 전분은 효소단백질 대비 13.2% (monomer), 13.5% (pentamer), 산화말토헥사오스는 9.7% (pentamer), 9.3% (monomer)로 나타났다.

• 미생물학회지
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• 제31권2호
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• pp.175-178
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• 1993

Alkaline .alpha.-amylase expressed in the transformant, Baciollus subtills MB809, containing alkaline amylase gene cloned from alkalophilic Bacillus sp. AL-8, was purified through for step separation processes. The purified alkaline .alpha.-amylase had molecular weight of app[roximately 59, 000 daltons on SDS-PAGE and Sephaex G-100 gel filtration. Amino acid sequence of terminal portion of the enzyme was analyzed with pure amylase eluted form the SDS-PAGE gel. N-terminal amino acid sequence of .alpha.-amylase was determined by the Edman degradation method and resulted in $NH_{2}$-ser-thr-ala-pro-ser-(ile)-lys-ala-gly-thr-(ile)-leu. For C-terminal amino acid sequencing, purified .alpha.-amylase was digested with carboxypuptidase A and B, and reverse-phase HPLC gradient elution system resulted in -thr-trp-pro-lys-COOH.

• BMB Reports
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• 제33권4호
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• pp.326-331
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• 2000

Acidic chitinases from the gizzards of a broiler were purified to homogeneity, using precipitation with $(NH_{4})_{2}SO_{4}$, ion exchanger chromatography, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The enzymes, GAC1 and GAC2, were purified 180- and 194- folds with a recovery of 4.9% and 2.7%, respectively. The molecular mass of GAC1 and GAC2 were 48.2 kDa and 57.8 kDa, respectively. Chromatofocusing resulted in a pI of 3.1 for both enzymes. The purified enzymes were endochitinases that were devoid of ${\beta}-N-acetylglucosaminidase$ and lysozyme activity. Kinetic studies using $[^3H]chitin$ indicate that GAC1 has a $K_m$ and $V_{max}$ of 1.97 mg/ml and 185 mg/mg protein/h, respectively. The GAC2 has a $K_m$ and $V_{max}$ of 0.42 mg/ml and 92.3 mg/mg protein/h, respectively at optimal pH and temperature (pH 5.0 and $60^{\circ}C$). When the pentamer and hexamer of N-acetylglucosamine (GlcNAc) were used as a substrate, the major product by GAC1 was the dimer of GlcNAc with a differential accumulation of the monomer and trimer, depending upon the substrate. However, the GAC2 produced the dimer and trimer in an equal quantity, regardless of the substrate used. The first 9 $NH_2-terminal$ amino acid residues of the purified gizzard chitinase GAC1 and GAC2 shared a 100% homology. The first 25 $NH_2-terminal$ amino acid residues of GAC1 also shared 55-60% homology with animal chitinases and some animal proteins, such as whey protein and oviduct-specific proteins. However, little homology was found with either microbial and plant chitinases, or egg white lysozyme.

• BMB Reports
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• 제43권11호
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• pp.726-731
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• 2010

We report the tissue-specific distribution of chitinolytic activity in Korean ginseng root and characterize two 31-kDa chitinolytic enzymes. These two enzymes (SBF1 and SBF2) were purified 70- and 81-fold with yields of 0.75 and 1.25%, respectively, and exhibited optimal pH and temperature ranges of 5.0-5.5 and 40-$50^{\circ}C$. With [$^3H$]-chitin as a substrate, $K_m$ and $V_{max}$ values of SBF1 were 4.6 mM and 220 mmol/mg-protein/h, respectively, while those of SBF2 were 7.14 mM and 287 mmol/mg-protein/h. The purified enzymes showed markedly less activity with p-nitrophenyl-N-acetylglucosaminide and fluorescent 4-methylumbelliferyl glycosides of D-N-acetylglucosamine oligomers than with [$^3H$]-chitin. End-product inhibition of both enzymes demonstrated that both are endochitinases with different N-acetylglucosaminidase activity. Furthermore, the $NH_2$-terminal sequence of SBF1 showed a high degree of homology with other plant chitinases whereas the $NH_2$-terminal amino acid of SBF2 was blocked.

• Parasites, Hosts and Diseases
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• 제38권2호
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• pp.107-110
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• 2000

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g. HSP70-NH2-terminal region, or rT.g. HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NHa-terminal region did not. These results suggest that T.g. HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.